Using display technologies to identify macrocyclic peptide antibiotics

Abstract

Antibiotic resistant bacterial infections are now a leading cause of global mortality. While drug resistance continues to spread, the clinical antibiotic pipeline has become bare. This discord has focused attention on developing new strategies for antimicrobial discovery. Natural macrocyclic peptide-based products have provided novel antibiotics and antibiotic scaffolds targeting several essential bacterial cell envelope processes, but discovery of such natural products remains a slow and inefficient process. Synthetic strategies employing peptide display technologies can quickly screen large libraries of macrocyclic sequences for specific target binding and general antibacterial potential providing alternative approaches for new antibiotic discovery. Here we review cell envelope processes that can be targeted with macrocyclic peptide therapeutics, outline important macrocyclic peptide display technologies, and discuss future strategies for both library design and screening.

Keywords: Antimicrobial discovery; Macrocyclic peptide; Peptide antibiotic; Peptide display.

Figure 1.. Gram-negative cell envelope drug targets.

Diagram of important gram-negative bacterial cell envelope processes with the outer and inner membranes (OM; IM) and peptidoglycan cell wall labelled. The Beta barrel assembly machinery (Bam; blue), lipopolysaccharide transport complex (Lpt; green), lipoprotein transport system (Lol; purple), Signal peptidases I and II (orange), and Disulfide bonding system (Dsb, yellow) are highlighted. Portions of lipid II and lipoprotein trafficking are also represented.

Figure 2.. Peptide cyclization strategies for mRNA display.

A) An mRNA library is attached to puromycin via a DNA linker on the 3’ end. The mRNA molecules are translated in vitro and covalently linked to the C-terminus of its coding peptide through puromycin at the end of translation. Following purification, reverse transcription generates the complementary DNA to prevent binding from the mRNA aptamer in the downstream screening. B) Crosslinking of cysteines with dibromo-xylene (DBX). C) Thioether bond formed by a cysteine and 2,3-didehydroalanine (Dha). D) Crosslinking of amine groups from the peptide N-terminus and a lysine sidechain by disuccinimidyl glutarate (DSG). E) Cyclization of an N-terminal Nα-chloroacyl-Phenylalanine and a cysteine side chain. The starting amino acid was recoded to Nα-chloroacyl-Phenylalanine which enabled the cyclization to happen spontaneously upon translation. F) mRNA display of a head-to-tail cyclized peptide. After translation, a series of reactions were taken out, resulting in: i) the N-terminal portion of the peptide (up to the Cysteine residue) being attached to the C-terminal portion of the peptide through side chains (light green and pink residues); ii) Native chemical ligation (NCL) for head to tail cyclization of the N-terminal portion of the peptide.

Figure 3.. Surface localized antimicrobial display of macrocyclic peptides.

Diagram of the surface localized antibacterial macrocyclic peptide display system. The display machinery is transported to the periplasm by a signal peptide which is cleaved and then is embedded into the outer membrane (OM) by encoding a fragment of E. coli OmpA. A long tether then leads to the variable region of the peptide library. Macrocyclic structure is potentiated through two cysteines (yellow) encoded on the terminal ends of the variable region potentiating a disulfide bond.